Reversal of diabetes in non-obese diabetic mice without spleen cell-derived beta cell regeneration

Autoimmune destruction of beta cells is the predominant cause of type 1 diabetes mellitus (T1DM) in humans and is modeled in non-obese diabetic (NOD) mice. Many therapeutic interventions prevent the development of T1DM in NOD mice, but few can induce its reversal once established. Intervention with Freund's complete adjuvant, semi-allogeneic splenocytes, and temporary islet transplantation has been reported to cure NOD mice of established T1DM. Using the same approach, we report here that this treatment cured 32% of NOD mice of established diabetes (>340 milligrams per deciliter blood glucose), although beta cells in these mice were not derived from donor splenocytes.     

Persistence of Mycoplasma synoviae in hens after two enrofloxacin treatments and detection of mutations in the parC gene

The ability of Mycoplasma synoviae, an avian pathogen, to persist despite fluoroquinolone treatments was investigated in hens. Groups of Mycoplasma-free hens were experimentally infected with the M. synoviae 317 strain and treated twice with enrofloxacin at the therapeutic dose. The results show that the two treatments did not have any influence on this strain of M. synoviae recovery from tracheal swabs. Mycoplasmas were isolated from tracheal swab cultures, but not from inner organs such as the liver or spleen, suggesting that this strain of M. synoviae was not able to cross the mucosal barrier to disseminate throughout the host. A significant increase of the resistance level to enrofloxacin of five re-isolated mycoplasma clones, was observed after the second treatment. This increase was associated in two clones to a Ser81-->Pro substitution, found in the ParC quinolone-resistance determining region (QRDR) of DNA topoisomerase IV. This is the first time that a mutation in a gene coding for topoisomerase IV is described in M. synoviae after in vivo enrofloxacin treatments in experimentally infected hens.     

Bilberry adulteration using the food dye amaranth

Vaccinium myrtillus or bilberry fruit is a commonly used herbal product. The usual method of determining the anthocyanin content is a single-wavelength spectrophotometric assay. Using this method, anthocyanin levels of two extracts were found to be 25% as claimed by the manufacturers. When high-performance liquid chromatography (HPLC) was used, however, one extract was found to contain 9% anthocyanins probably not derived from V. myrtillus but from an adulterant. This adulterant was subsequently identified, using HPLC, mass spectroscopy, and nuclear magnetic resonance, as amaranth, that is, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt-a synthetic dark red sulfonic acid based naphthylazo dye. As described in this study, if deliberate adulteration occurs in an extract, a single-wavelength spectrophotometric assay is inadequate to accurately determine the levels of compounds such as anthocyanins. Detection of deliberate adulteration in commercial samples thus requires the use of alternative, more sophisticated, methods of analysis such as HPLC with photodiode array detection as a minimum.     

Isobaric labeling approach to the tracking and relative quantitation of peptide damage at the primary structural level

Protein oxidative damage lies behind skin and hair degradation and the deterioration of protein-based products, such as wool and meat, in addition to a range of serious health problems. Effective strategies to ameliorate degenerative processes require detailed fundamental knowledge of the chemistry at the molecular level, including specific residue-level products and their relative abundance. This paper presents a new means of tracking damage-induced side-chain modification in peptides using a novel application for isobaric label quantification. Following exposure to heat and UVA and UVB irradiation, tryptophan and tyrosine damage products in synthetic peptides were characterized and tracked using ESI-MS/MS and iTRAQ labeling-based relative quantification. An in-depth degradation profile of these peptides was generated, enabling the formation of even low-abundance single-residue-level modifications to be sensitively monitored. The development of this novel approach to profiling and tracking residue-level protein damage offers significant potential for application in the development and validation of protein protection treatments.     

Sexual Transmission of Somatic Embryogenesis in Dactylis glomerate*

A Dactylis glomerata L. genotype that produces somatic embryos in vitro was tested for the ability to sexually transmit the embryogenic trait. Reciprocal crosses were performed between the embryogenic and two non-embryogenic genotypes. Leaf segments from 69 F₁ plants were cultured on Schenk and Hildebrandt medium amended with 30 μM 3,6-dichloro-o-anisic acid (dicamba). Somatic embryogenesis was expressed in 39 of the F₁ plants. The embryogenic parent was female for 18 of these plants and male for the other 21. The 39:30 ratio of embryogenic: non-embryogenic fits an expected 1:1 for tetrasomic inheritance of a dominant nuclear gene.     

Changes in the activity and abundance of the soil microbial community in response to the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP)

Purpose

The application of organic and inorganic fertilizers to soil can result in increased gaseous emissions, such as NH₃, N₂O, CO₂, and CH₄, as well as nitrate leaching, contributing to climate warming and ground and surface water pollution, particularly in regions with hot climates, where high temperatures and high soil nitrification rates often occur. The use of nitrification inhibitors (NIs) has been shown to effectively decrease nitrogen (N) losses from the soil-plant system.

Materials and methods

Non-disruptive laboratory incubation experiments were conducted to assess the extent to which temperature (20 and 30 °C) and nutrient source (mineral and organic fertilizers) influence the rate of carbon (C)- and N-related microbial processes in soil in response to the NI 3,4-dimethylpyrazole phosphate (DMPP). Furthermore, short-term changes in the ability of microbes to degrade C substrates were evaluated in disruptive soil microcosms using microbial community-level physiological profiling and the abundance of the bacterial 16S rRNA gene as a measure of total bacterial population size.

Results and discussion

DMPP reduced net nitrification after 2 and 4 weeks of incubation at 30 and 20 °C by an average of 78.3 and 84.5 %, respectively, and with similar dynamics for mineral or organic fertilization. The addition of labile organic matter with cattle effluent led to a rapid increase in C mineralization that was significantly reduced by DMPP at both temperatures, whereas no changes could be detected after the addition of mineral fertilizer. The culturable heterotrophic microorganisms showed metabolic diversification in the oxidation of C sources, with organic fertilizer playing a major role in the substrate utilization patterns during the first week of incubation and the DMPP effects prevailing from day 14 until day 28. Furthermore, the copy number of the bacterial 16S rRNA gene was reduced by the application of DMPP and organic fertilizer after 28 days.

Conclusions

Our results show the marked efficiency of DMPP as an NI at elevated temperatures of incubation and when associated with both mineral and organic fertilization, providing support for its use as a tool to mitigate N losses in Mediterranean ecosystems. However, we also observed impaired C respiration rates and bacterial abundances, as well as shifts in community-level physiological profiles in soil, possibly indicating a short-term effect of DMPP and organic fertilizers on non-target C-related processes and microorganisms.

     

Amplification facilitators and multiplex PCR: Tools to overcome PCR-inhibition in DNA-gut-content analysis of soil-living invertebrates

Polymerase chain reaction (PCR) can be used to detect prey within the gut contents of predators and allows specific trophic interactions to be studied among soil-dwelling invertebrates which cannot be examined by other approaches. PCR-inhibitory substances, however, are commonly found in DNA prepared from soil organisms or from biological material contaminated with soil. This can lead to false-negative results and the risk of not detecting trophic connections or of underestimating predation rates in field studies. In the present study, we developed mitochondrial DNA markers to detect Amphimallon solstitiale (Coleoptera: Scarabaeidae) in the gut contents of invertebrate predators. Larvae of A. solstitiale can cause serious damage in grasslands, field crops, and forests by feeding on roots. Adequate methodologies to study predation on these pests are lacking, and their invertebrate predator guild is, therefore, barely known. To test the new molecular markers for prey detection, larvae and eggs of A. solstitiale were fed to Poecilus versicolor larvae (Coleoptera: Carabidae), which are abundant below-ground predators in grassland ecosystems. Unfortunately, even when specific DNA extraction and purification methods were used, DNA extracts from predators were of poor quality and not amplifiable by PCR; this yielded false-negative results and a dramatically lower prey-detection rate. We overcame PCR-inhibition by applying ?1.28 μg μl⁻¹ bovine serum albumin to the PCR reaction mix. This enabled us to detect A. solstitiale DNA within fed carabid larvae up to 48 and 40 h post-feeding for 127 and 463 bp sized DNA fragments, respectively. When single A. solstitiale eggs were consumed by the carabid larvae, predation could be verified in 100% of the predators within the first 8 h of digestion; some carabid larvae even tested positive 32 h after feeding. Moreover, by multiplexing primers targeting both prey and predator, we were able to simultaneously screen for prey consumption and check for co-purified PCR inhibitors. Sensitivity in prey detection was not reduced compared to singleplex PCR. We recommend the multiplex approach because it considerably reduces time and costs compared to singleplex assays. We also show that multiplex PCR not only detects specific prey, but also can identify the predator itself. This allows the identification of taxa which are difficult or not identifiable based on morphological characters, such as soil-dwelling predatory beetle larvae.     

The effect of provenance on the establishment and performance of Lotus corniculatus L. in a re-creation environment

Field trials investigated the effect of provenance on the establishment of Lotus corniculatus at a limestone quarry re-creation site. Cuttings were collected from two habitats (calcareous and non-calcareous) within 15 regions in the British Isles. Two ramets of each plant were propagated and planted in untreated (bare clay substrate) and treated plots (with topsoil). We investigated the effect of geographical and ecological distance on plant survival, size and fecundity. Local plants had greatest survival on the treated plots although this was not the case on the untreated plots where other provenances also performed well. In addition, there was a significant, albeit very weak, negative relationship between survival and geographical distance on the treated plots and plant size and fecundity on the untreated plots. In contrast, the effect of ecological distance was only significant for plant size on the untreated plot; plants from more ecologically distant populations were larger and more fecund. This result was unexpected and may reflect adaptations amongst chalk grassland ecotypes (e.g. dwarfing) to extreme conditions (e.g. drought). Cyanogenesis, which has previously been linked to aspects of plant fitness, was not shown to have an effect on plant performance. In conclusion, this study suggests that there are regional differences between populations of L. corniculatus which may affect the performance of plants in re-creation schemes. Ecological provenance may also influence the fitness of plants when translocated. However, there was no evidence to support a consistent home-site advantage of local genotypes. Further long-term studies on a range of species are necessary.     

Evaluating N/N and C/C isotope ratio analysis to investigate trophic relationships of elaterid larvae (Coleoptera: Elateridae)

Carbon and nitrogen isotope ratios in consumer tissues can be used to analyse the diet and trophic level of soil animals. However, life history traits may significantly influence stable isotope patterns. We evaluated in a series of experiments how stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) at natural abundance can be used to study the diet and trophic position of long-lived macro-invertebrates, elaterid larvae, which are major below-ground herbivores. Small, but significant differences in δ¹³C signatures were found between the larvaes’ anterior and posterior body segments, whereas exuvia reflected the body's overall isotopic composition. The species-specific trophic shift (±SE) in δ¹⁵N for Agriotes obscurus and Agriotes sputator (1.62±0.24‰ and 1.08±0.27‰, respectively) was significantly lower than “mean enrichment estimates” reported in the literature, showing the limited applicability of such generalised estimates in studies of invertebrate trophic ecology. To avoid false-positive assignments to two trophic levels due to variation in δ¹⁵N values, a minimum sample size of three and five individuals for A. obscurus and A. sputator, respectively, was needed to reduce this risk to below α=5%. Keeping elaterid larvae for up to 128 days without food did not affect their isotopic signatures, in contrast to previous studies on starving animals. Switching wireworms to isotopically different diets induced changes in their isotopic signatures within 2 weeks. Changes, however, were significant only when the isotopic difference between diets was large. We conclude that experimental studies evaluating how specific life history traits affect stable isotope signatures in consumers have to precede any interpretation of stable isotope data gathered in the field.